Therapy for hepatitis C virus (HCV) infection has advanced rapidly with the recent approval of several direct-acting antivirals. However, most of the DAAs in clinical use or clinical trials target the same stage of HCV replication cycle and are associated with rapid emergence of drug-resistant viral mutations. In addition, different HCV genotypes and clinical conditions may also require adjustment of treatment regimen. Therefore, there is still an ongoing need to develop new HCV inhibitors that target different stages of the HCV replication cycle, such as entry and assembly. A novel aryloxazole-based HCV entry inhibitor was identified previously through a quantitative phenotypic high-throughput screen. Structureactivity relationship (SAR) optimization yielded the compound fluoxazolevir with the improved efficacy, cytotoxicity and in vitro absorption-distribution-metabolism-excretion properties as a lead candidate for preclinical development. Here we characterize its mechanism of action, in vitro efficacy against various HCV genotypes, synergy with other HCV drugs, pharmacokinetics and in vivo efficacy in a HCV-infected humanized mouse model. Time-of-addition experiments in cell-based infection and membrane fusion assays indicate that this compound targets the viral fusion step of the HCV life cycle. Fluoxazolevir showed antiviral synergy with FDA-approved HCV drugs in vitro and effectively inhibited all chimeric HCV genotypes with varying EC50 values. Pharmacokinetic studies showed preferential localization of fluoxazolevir in the liver with long t1/2 values (1737 h) for PO and IV routes. In human hepatocyte-engrafted Alb-uPA/Scid mice infected with HCV genotype 1b, 2a or 3, fluoxazolevir monotherapy for 4 weeks showed a 2-log reduction in viral titer without evidence of drug resistance. In combination with daclatasvir (NS5A inhibitor), fluoxazolevir led to a sustained virologic response after 4 weeks of treatment against HCV genotypes 1b and 3. In combination with glecaprevir (NS3/4A protease inhibitor) and pibrentasvir (NS5A inhibitor), fluoxazolevir could achieve sustained virologic response in mice infected with a multidrug-resistant HCV strain. Fluoxazolevir is a promising preclinical lead in the next generation of combination drug cocktails for HCV treatment. From the same HTS, we also identified chlorcyclizine HCl (CCZ), an over-the-counter drug for allergy symptoms, and related compounds as potent inhibitors of HCV infection. CCZ is a HCV inhibitor targeting late viral entry. Here we identify the molecular target of CCZ. In CCZ resistance-associated strains generated by an in vitro selection assay, six mutations were found in the E1 glycoprotein with 5 in the fusion loop. These mutations conferred resistance when introduced into the HCV genome. CCZ blocked HCV membrane fusion and the mutants acquired resistance to CCZ at this step. UV cross-linking of HCV-infected cells or recombinant HCV E1/E2 with CCZ-diazirine-biotin identified cross-linked E1 glycoprotein. Mass spectrometry demonstrated that CCZ cross-linked with E1 sequences adjacent to the fusion loop. Molecular simulations revealed that CCZ forms extensive interactions with the fusion loop. These results suggest that CCZ inhibits HCV infection by binding to the fusion loop of E1.